Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378, USA.
FEBS Lett. 2010 Dec 1;584(23):4725-30. doi: 10.1016/j.febslet.2010.10.060. Epub 2010 Nov 5.
The factor inhibiting HIF-1 (FIH-1) hydroxylates many ankyrin repeat-containing proteins including IκBα. It is widely speculated that hydroxylation of IκBα has functional consequences, but the effects of hydroxylation have not been demonstrated. We prepared hydroxylated IκBα and compared it to the unhydroxylated protein. Urea denaturation and amide H/D exchange experiments showed no change in the "foldedness" upon hydroxylation. Surface plasmon resonance measurements of binding to NFκB showed no difference in the NFκB binding kinetics or thermodynamics. Ubiquitin-independent proteasomal degradation experiments showed no difference in the half-life of the protein. Thus, it appears that hydroxylation of IκBα by FIH-1 is inconsequential, at least for the functions we could assay in vitro.
因子抑制 HIF-1（FIH-1）羟化许多含有锚蛋白重复序列的蛋白质，包括 IκBα。人们普遍推测 IκBα的羟化具有功能后果，但羟化的影响尚未得到证实。我们制备了羟化的 IκBα并将其与未羟化的蛋白质进行了比较。尿素变性和酰胺 H/D 交换实验表明，羟化后蛋白质的“折叠”没有变化。表面等离子体共振测量表明，NFκB 的结合动力学或热力学没有差异。非依赖于泛素的蛋白酶体降解实验表明，蛋白质的半衰期没有差异。因此，FIH-1 对 IκBα的羟化似乎没有影响，至少对于我们在体外进行的功能测定而言是这样。
